The abundant class III chitinase homolog in young developing banana fruits behaves as a transient vegetative storage protein and most probably serves as an important supply of amino acids for the synthesis of ripening-associated proteins

Abstract

Analyses of the protein content and composition revealed dramatic changes in gene expression during in situ banana (Musa spp.) fruit formation/ripening. The total banana protein content rapidly increases during the first 60 to 70 d, but remains constant for the rest of fruit formation/ripening. During the phase of rapid protein accumulation, an inactive homolog of class III chitinases accounts for up to 40% (w/v) of the total protein. Concomitant with the arrest of net protein accumulation, the chitinase-related protein (CRP) progressively decreases and several novel proteins appear in the electropherograms. Hence, CRP behaves as a fruit-specific vegetative storage protein that accumulates during early fruit formation and serves as a source of amino acids for the synthesis of ripening-associated proteins. Analyses of individual proteins revealed that a thaumatin-like protein, a beta-1,3-glucanase, a class I chitinase, and a mannose-binding lectin are the most abundant ripening-associated proteins. Because during the ripening of prematurely harvested bananas, similar changes take place as in the in situ ripening bananas, CRP present in immature fruits is a sufficient source of amino acids for a quasi-normal synthesis of ripening-associated proteins. However, it is evident that the conversion of CRP in ripening-associated proteins takes place at an accelerated rate, especially when climacteric ripening is induced by ethylene. The present report also includes a discussion of the accumulation of the major banana allergens and the identification of suitable promoters for the production of vaccines in transgenic bananas.

Figure 1

A, Changes in size and weight during in situ development and ripening of banana fruits. Open arrows numbered 1 through 7 indicate at which stage the seven different PCI were reached. <1 means that the fruits did not yet reach the stage corresponding to PCI 1, whereas >7 refers to overripe bananas (peel color turning from brown to black). At 156 DPA (indicated by the black arrow), hands were cut and ripening continued under laboratory conditions. The time of harvest (70 DPA) of the immature fruits destined for the study of the ex planta ripening is indicated by a triangle. B, Changes in fruit, pulp, and peel weight of ex planta-ripening bananas in the absence (Co) and presence of ethephon (ETH). White and black arrows indicate when the seven different PCI were reached in control and ethephon-treated bananas, respectively.

Figure 2

SDS-PAGE of crude extracts from the pulp (A) and peel (B) from in situ-developing/ripening banana fruits. A mixture of BanGase (31 kD), BanTLP (20 kD), and BanLec (15 kD) was loaded in lane BP. Molecular mass reference proteins (lane R) were lysozyme (14 kD), soybean (Glycine max) trypsin inhibitor (20 kD), carbonic anhydrase (30 kD), ovalbumin (43 kD), bovine serum albumin (67 kD), and phosphorylase b (94 kD). A, Lanes a through r refer to samples of banana pulp taken 24, 30, 39, 48, 56, 66, 80, 92, 106, 120, 135, 147, 152, 156, 160, 164, 168, and 171 DPA, respectively. B, Lanes a through p show samples of banana peel taken after 24, 30, 39, 48, 56, 66, 80, 92, 106, 120, 135, 147, 152, 156, 160, and 164 DPA.

Figure 3

Changes in total protein, CRP, and BanTLP concentration (A and D), total protein, CRP, and BanTLP content (B and E), and BanGase, BanChi, and BanLec content (C and F) in the pulp (A–C) and peel (D–F) from in situ-developing/ripening banana fruits.

Figure 4

Changes in total protein (A), CRP (B), BanTLP (C), BanGase (D), BanChi (E), and BanLec (F) concentration in the pulp and peel of ex planta-ripening bananas in the absence (Co) and presence of ethephon (ETH).

Figure 5

SDS-PAGE of crude extracts from pulp and peel of bananas ripening ex planta in the absence (A) and presence (B) of ethephon. A, Lanes a through g and h through n show crude extracts from pulp and peel of control bananas, respectively. Samples were taken 0, 4, 8, 12, 16, 20, and 24 d after detachment of the bananas from the tree. B, Lanes a through c and d through f show the protein pattern in pulp and peel of ethephon-treated bananas sampled 0, 4, and 8 d after detachment of the bananas. Lanes R and BP were as indicated in Figure 2.